We propose to use the inhibitory synapse of the crayfish neuromuscular junction to investigate mechanisms underlying synaptic plasticity with a presynaptic voltage control method. This method allows us to use presynaptic steps of defined duration and amplitude, rather than action potentials, to activate transmitter release. With this method, we have been able to separate two mechanisms that increase transmitter output downstream of calcium influx. The first one accelerates transmitter release kinetics and the second mechanism increases the number of available vesicles. We have shown that synaptic facilitation only utilizes the former while okadaic acid (OA) and serotonin can activate both mechanisms. In addition, the accelerated release kinetics is accompanied by a reduction in the calcium cooperativity of transmitter release. These observations forrn the bases of the pro osed studies outlined here. We propose to use the presynaptic voltage control method to investigate physiological mechanisms underlying long term facilitation (LTF) and second messenger, protein kinase A and C (PKA and PKC). mediated synaptic enhancement. Our preliminary data show that LTF increases the number of available vesicles without changing release kinetics. Results from this study should provide new insights to the physiological conditions under which a specific presynaptic mechanism can be selectively activated. In addition, since it has been shown that the kinase A pathway underlies LTF, we also propose to perform kinetics analysis of transmitter release mediated by PKA and PKC. Our working hypothesis is that PKA and PKC may modulate vesicle availability and release kinetics, respectively. This series of studies should provide new insights to the relative importance of individual kinase pathways on different forms of synaptic plasticity. We also propose to use calcium imaging techniques to measure changes in calcium cooperativity associated with different forms of synaptic plasticity. Since we have shown that accelerated release kinetics during facilitation is accompanied by a reduction in cooperativity, we sought to measure the cooperativity, with imaging techniques, under a different condition where an obvious acceleration in release kinetics has been shown. Our preliminary data show that calcium cooperativity decreases in OA. We propose to examine calcium cooperativity during LTF, in OA, and in PKA and PKC activated synaptic enhancement. Our working hypothesis is that decreased cooperativity will only be observed under conditions that accelerate release kinetics and not under those that increase the vesicle availability. Changes in calcium cooperativity add a new facet to our thinking of synaptic plasticity.
